Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
  • A61K47/557—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
  • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

• the present invention relates to new nanoassembled complexes (also hereinafter known as nanocomplexes or nanoassemblies) and more specifically to nanoassemblies of nucleic acids, avidin and polymers, to their use in the biotechnological field and nanomedicine and to their preparation.
• nanocomplexes or nanoassemblies also hereinafter known as nanocomplexes or nanoassemblies
• nanoassemblies of nucleic acids, avidin and polymers
• Avidin is a tetrameric glycoprotein known mainly for its ability to bind to four molecules of biotin with very high affinity (K d ⁇ 10 "15 M). From the practical viewpoint, the avidin property of a high and multiple affinity for biotin forms the basis for its use as a molecular instrument in a large number of biotechnological applications (avidin-biotin technology) (Wilchek M and Bayer EA, Analytical Biochemistry. 1988, 171 : 1 -32; Wilchek M and Bayer EA, Methods Enzymol. 1990, 184: 14-45). In respect of this property, avidin can serve as a molecular bridge to then stably link together different biological or chemical units, provided that these latter are covalently bound to one molecule of biotin.
• avidin-biotin technology is for analytical purposes, more precisely for detection and quantification systems which are usually based on the ability to link an antibody, or any other molecule having high affinity towards the analyte (ligand/antigen), to a marker system (a fluorophore, an enzyme able to emit light/colour, a radionuclide etc.); other applications include surface functionalization with specific chemical/biochemical entities, being a procedure which is often conducted by using the molecular bridge formed from the avidin-biotin complex; another application is for targeting drugs or diagnostic elements, administered by parenteral means, towards specific sites in the body (Goldenberg DM, Sharkey RM, Paganelli G, Barbet J, and Chatal JF, J. Clin. Oncol. 2006, 24: 823-834).
• This increased capability can be achieved by joining together several avidin molecules into a single unit (defined herein as a poly-avidin unit).
• the literature describes various technological approaches for obtaining said poly- avidin nuclei.
• the strategies commonly adopted and currently available are based on coating the surfaces of micro- or nano-spheres (consisting of different polymers such as polystyrene or metals, such as gold) with several avidin molecules, or on the chemical "polymerization" of avidin by covalent crosslinking.
• Strategies currently available for forming poly-avidin units are hence based either on chemical synthesis processes aimed at the formation of covalent bonds between avidin units, or on non-specific adsorption processes which lead to avidin molecules adhering to the surfaces of polymer or metal nuclei.
• the poly-avidins thus obtained are always characterized by: a) a certain degree of polydispersivity depending on the method for obtaining them: b) a partial loss of avidin activity.
• inactivation of avidin translates into a reduced capacity for binding with biotin (and hence with any other biotinylated ligand), whereas polydispersivity translates into products whose properties are statistically defined and are hence not highly defined.
• poly-avidins obtained by means of the aforesaid methods are either not of natural origin or are not always biocompatible and therefore potentially toxic.
• the poly- avidins obtained by these methods can thus present toxicological risks related to the elements comprising them and this limits their applicability in pharmaceutical/diagnostic environments when in vivo contact of the avidin assembly with human or animal tissue is envisaged.
• nanoparticulate structures of toroid or rod shape can be obtained, in which a single nucleic acid molecule is surrounded by several avidin molecules.
• the nanoassemblies are poorly polydispersed and their size depends solely on the type and length of the nucleic acid used.
• these latter arrangements which are already described in the literature (Morpurgo M et al. 2004 ref. cit), are stable and isolatable in aqueous salt-free solution; in the presence of electrolytes they undergo a rapid process of aggregation subsequent to which polydispersed macro-aggregates are again obtained but actually unusable for practical purposes.
• the avidin/biotin system comprises biorecognition reactions in saline aqueous environment
• the avidin and nucleic acid complexes described above have no practical use because they are unable to exist as discrete and stable entities under the required buffered conditions.
• aggregation is a general problem common to many small sized particles, particularly when they fall within the colloidal range ( ⁇ 1 micron - nanoparticles).
• Aggregation depends on particle surface characteristics (charge type and density, hydrophobicity, hydrophilicity, etc.) and on the type of medium in which they are suspended (inorganic solvent, aqueous solvent, type of buffer, ionic strength, pH, etc.); various technical solutions can be employed to avoid or slow down aggregation.
• hydrophilic polymers which are covalently bound or adsorbed onto the particle surface so as to partially or completely conceal it from the surrounding environment. A steric hindrance and an enthalpic gain are thus created which prevent the particles from interacting irreversibly with each other.
• hydrophilic polymers are used to protect the surface of liposomal nanoparticles (Cattel L, Ceruti M, et al. Tumori, 2003, 89:237-249) used as carriers of antitumour drugs to be administered by parenteral means.
• hydrophilic polymers in preventing non-specific interactions between different surfaces (and hence also between nanoparticles) to which they are attached is related to two parameters: a) polymer chain length and b) grafting density (Jeon Sl, Lee JH et al. J. Colloidal and Interface ScL 1991 , 142: 149-158; Jeon SI and Andrade JD J. Colloidal and Interface Sci. 1991 , 142: 159-166; Sofia SJ, Premnath V et al. Macromolecules 1998, 31 : 5059-5070). For each system therefore, the same efficacy of aggregation prevention is achievable by varying one, or the other or both the aforesaid parameters.
• each system whether surface or nanoparticulate, is characterised by distinctive properties (chemical, angle of curvature, etc.) and so the efficacy of surface protection must be calibrated each time in order to optimize the effects.
• various parameters are taken into account during optimization and include type of polymer, its length and attachment density, and not least, the grafting method (Owens DE and Peppas NA Int. J. Pharm. 2006, 307: 93-102). Consequently, the results obtained with a determined particle system are not directly transferable to another one and as such, the information already described in the literature is not directly applicable to nanoparticulate systems consisting of avidin and nucleic acids.
• One aspect of the present invention is to obtain nanoparticles consisting of nucleic acids and avidin which are stable in an aqueous/saline environment.
• a further aspect of the present invention is that said stable systems are able to recognize other biotinylated elements, in that they themselves possess pharmacological activity, or are able to recognize third elements (for example a receptor) or are able to generate signals by themselves or in combination with other reagents in solution (for example fluorescence, colour, radioactivity, photons.) Summary
• the nanoassembled complexes provided by the inventors fulfil the aforementioned purposes, as they allow the previously reported drawbacks derived from the known technologies of the art to be overcome.
• the obtained nanoassembled complexes are highly defined from the qualitative and quantitative composition viewpoint and stable even in the presence of electrolytes.
• the object of the present invention are nanoassembled complexes comprising a nucleus obtained by means of high affinity interaction between one or more avidin units and one or more nucleic acid molecules, wherein said nucleus is stabilized by a biotinylated surface protecting agent, represented by the general formula (I)
• - NB are the single nuclobases of a single or double stranded nucleic acid; - Av is an avidin unit;
• PA is a polymer unit having at least one or two functionalizable residues of which one binds, by a covalent bond either directly or through a spacer X, to a biotin residue B by means of carboxyl functional group thereof;
• - n is a number varying from 16 to 10,000,000;
• - b is a number varying from 1 to 128.
• nanoassembled complexes of the invention are in the form of nanoparticles which are another object of the invention.
• a further object of the invention is the use of nanoassembled complexes of formula (I) as means vitro and in vivo diagnostics, in the field of nanomedicine for targeting and concentrating bioactive molecules towards specific sites in the body, in the field of nanotechnology in general for the localization of molecules onto surfaces, and in any application (biomedical and engineering) that requires a co- localization of several chemical or biological functions of varying natures on a central core, being in its turn present in colloidal suspension or localized onto a surface.
• a still further object of the invention is a method for preparing the nanoassembled complexes of general formula (I).
• FIG. 1 the figure shows the size distribution (INTENSITY-weighted-GAUSSIAN Analysis) of the particles of the nanoassembled complexes
• A) Av-pEGFP 3 (sample 1 of examples 1 and 2); B) Av-pEGFP 3-B-X a -PAi,-IV-30 (sample 26 of example 2); C) Av-GenNB 2-B (sample 31 of example 4); D) Av-GenNB 2-B-X a - PAi, IV-30 (sample 35 of example 4).
• Figure 2 the figure shows the kinetics of aggregation in a buffered solution of the different nanoassembled complexes of example 2 as a function of the type of B-
• Biotin Binding Sites equal to 0 (•), 20 (O), 30 ( ⁇ ), 40 ( ), 50 ( ⁇ ) 1 60 ( ⁇ ) %
• B B- X 3 -PAi, Na, % of occupied BBS equal to 0 (•), 20 (O), 30 ( ⁇ ), 40 ( ), 50 ( ⁇ ), 60 ( ⁇ ) %
• C B-X 3 -PAi, Nb, % of occupied BBS equal to 0 (•), 20 (O), 30 ( ⁇ ), 40 ( ), 50 ( ⁇ ), 60 ( ⁇ ) %
• D B-X 3 -PAi, III, % of occupied BBS equal to 0 (•), 20 (O), 30 ( ⁇ ), 40 ( ), 50 ( ⁇ ), 60 ( ⁇ ) %
• Figure 3 the figure shows fluorescent microscope images of membranes used in an assay, with dot blot fluorescent detection, comparing avidin in monomeric form and in nanocomplexed form with nucleic acid. Incubation was carried out using avidin-biotin-Alexa solutions at 1.3 ⁇ g/ml.
• A1 monomeric avidin (sample 38 example 5);
• A2 Av-pEGFP 1.5 B-X 3 -PAi, IV-25 (sample 39 example 5);
• A3 Av- pEGFP 0.75 B-X 3 -PAi, IV-25 (sample 40 example 5).
• Figure 4 the figure shows fluorescent microscope images of membranes used in a further assay, with dot blot fluorescent detection, comparing avidin in monomeric from and in nanocomplexed form with nucleic acid. Incubation was carried out using avidin-biotin-Alexa solutions at 5 ⁇ g/ml in monomeric form (sample 38 of examples 5 and 6) and in nanoassembly form (sample 41 example 6).
• Figure 5 the figure shows the comparison of detecting efficiency of avidin in a monomeric (o) ad nanocomplexed (•) form with nucleic acid, in a dot blot with enzyme (HRP)-linked detection system.
• nanoassembled complexes of the present invention discrete nanoparticles are obtained which are stabilized against risks of: a) aggregation in aqueous saline environments and b) non-specific interactions with other molecules in solution, by virtue of the presence of protective elements on their surface.
• Said protective elements are themselves present on the particle surfaces in controlled and highly defined quantities. Moreover, surface protection according to the preparative method developed by the inventors takes place without destroying the nucleic acid-avidin self-assembled complex and without modifying the total capability of assembled avidins for binding to biotin (i.e. without modifying biotin binding sites).
• the size of these nanoparticles can be established from the length of the nucleic acid which is the assembling nucleus of more avidin units, and accordingly, particles characterized by different sizes and different charges on the avidin can be obtained by suitably varying the size of the nucleating nucleic acid (NA).
• NA nucleating nucleic acid
• the characteristics of said particles are precisely defined and their properties can be modulated by the user by varying: a) the type and size of the nucleating NA; b) the ratio between avidin and nucleic acid bases; c) the nature and quantity of the protecting agent present on the surface.
• the compounds object of the same are nanoassembled complexes comprising a nucleus obtained by nucleation secondary to a high affinity interaction of several avidin units onto one or more nucleic acid molecules, and stabilized by a biotinylated surface protecting agent, represented by the general formula (I)
• NB n AVy(B-X 3 -PA ⁇ z (I) wherein: - NB are the single nuclobases of a single or double stranded nucleic acid;
• - Av is an avidin unit
• PA is a polymer unit having at least one or two functionalizable residues of which one binds, by a covalent bond either directly or through a spacer X, to a biotin residue B by means of its carboxyl functional group;
• - a is a number varying from 0 to 50 and is preferably comprised from 0 to 10;
• - b is a number varying from 1 to 128
• the nanocomplexes of the invention can bind additional biotinylated compounds, different from the protecting agent, onto said binding sites.
• NB means a nucleic acid consisting of a number of nucleobases
• n n varying from 16 and 10,000,000, referring to the total number of bases, irrespective of whether the nucleic acid is single or double stranded.
• the nucleic acid consists of a base number varying from 30 to
• the base number is from 3,000 to 50,000.
• nucleic acid refers equally to: i) any sequence of a single stranded (ss) or double stranded (ds) deoxyribonucleic acid (DNA) polymer; ii) any sequence of a ribonucleic acid (RNA) polymer in single stranded form or hybridized with a RNA or a complementary DNA chain; iii) a sequence, in accordance with the above points, in which a part of or all the bases have been chemically modified.
• the usable nucleic acid for the nanoassembled complexes of formula (I) can be in linear or circular form, in a relaxed, coiled or supercoiled state.
• avidin is defined as being derived from chicken eggs or another similar source (eggs of birds in general) or from recombinant technology, either in glycosylated or deglycosylated form. Also included are other chemically or genetically modified avidin forms, provided they can assemble onto a single or double stranded nucleic acid as previously established. In view of the relationship between the number n of NB and the number y of avidin units self-assembling onto the nucleic acid, y is preferably comprised from (0.0001 )*n to (0.0357)'/7 and more preferably comprised from (0.01 )*n to (0.0357)'/7.
• PA means preferably a linear unit of a hydrophilic polymer of any molecular weight capable of binding to biotin by a covalent bond, either directly or through a spacer X, by means of the biotin carboxyl group. If PA has two functionizable residues, the second of said residues is free or protected by protecting groups known to an expert of the art, for example a methoxyl group.
• PA represents a hydrophilic polymer consisting of several polymer units, these latter are joined together by a further ligand having a number of functionalites equal to or greater than 3 (> 3) of which one binds to the spacer X or to biotin B and the remaining other functional groups bind to the polymer units PA;
• Y, Y' being the same or different from each other are -COO-; -NH -;-O-; SO 2 -; -S-; -SO-; -CO-; -COS-; -NH-CO-; -NH-CO-; HN-SO-NH- ;
• R can be an alkyl, an alkenyl, an alkinyl, a cycloalkyl, or an aryl with a carbon atom number comprised from 1 to 20 and preferably from 5 to 20, also optionally substituted.
• the bond between the spacer X and biotin B and that between the spacer X and the hydrophilic polymer PA can be indiscriminately an amide bond, an amino bond, a carbamide bond, an ester bond, a ketone bond, an ether bond, a thioester bond, a thioether bond, an urea bond, a thiourea, sulphonic or sulphoxide bond.
• units, z is comprised from (0.02)*y to [AYy, and preferably is comprised from (0.4)*yto (4)*y.
• the polymer units PA are biocompatible and preferably hydrophilic polymers and are known polymers (Owens DE and Peppas NA 2006 ref. cit.) in which the polymer unit PA has a molecular weight preferably comprised from 400 to 40,000 and more preferably from 1 ,000 to 20,000.
• Said polymer units are preferably selected from the group consisting of polyethylene oxide or polyethylene glycol (PEO or PEG) also optionally substituted, a copolymer of polyoxyethylene and polyoxypropylene (PEO-PPO), polyvinylpyrrolidone (PVP), polyacryloylmorpholine (PacM), a polyoxamine, a polylactide (PLA), a polyglycolide (PLG), a copolymer of lactic acid and glycolic acid (PLGA). More preferably the polymer PA is a substituted polyoxyethylene (PEO) and is therefore characterized by the following formula (III):
• R 1 , R 2 , R 3 and R 4 can be independently equal to hydrogen, alkyl, cycloalkyl, aryl, alkenyl, alkinyl, alcoxyl, thioalkoxy, aryloxy and thioaryloxy m is an integer from 2 to 900.
• the polymer consists of several polymer units, and these are bound together by a polyfunctional ligand with functionality equal to or greater than 3 (> 3), said ligand can be lysine, glutamic acid, aspartic acid, cysteine, a dendrimer.
• dendrimer means a symmetrical macromolecular compound consisting of branches repeated around a central core consisting of a smaller molecule or a polymeric nucleus.
• the functional groups present outside the dendrimer, whose number depends on its number of branches, are themselves functionalizable with other molecules including, for example, PA polymers.
• the polymer unit PA can further covalently bind, through a second free functional group, to a compound suitable for the uses pursued with the nanoassembled complex, and in particular compounds selected from ligands, sugars, chromophores or fluorophores, drugs, chelating agents for radionuclides, peptides, antibodies, proteins, enzymes and the like.
• the preparation of the nanoassembled complexes of the invention comprises three successive steps in aqueous solutions: in the first step nanoparticles consisting of only avidin and nucleic acid are obtained, constituting the central nucleus of the complexes of the invention.
• the two subsequent steps comprise optionally preparing the biotinylated surface protecting agent B-X 3 -PAi, but mainly adding said surface protecting agent to the nucleic acid-avidin nanoparticles obtained in the first step. Therefore, the method for preparing the nanoassembled complexes of general formula NB ⁇ Av y (B-X a -PAi,) z (I) comprises at least the steps of: a) preparing the self-assembled primary nucleus NB ⁇ Av y by mixing avidin Av with nucleic acid in predefined stoichiometric molar ratios of avidin to nucleobases; b) mixing the biotinylated surface protecting agent B-X 3 -PAi, with the previously obtained primary nucleus.
• preparation of the nanoassemblies of the invention can also comprise preparation of the biotinylated surface protecting agent B-X 3 -PAi, . .
• the first step is undertaken by mixing, under stirring, the solutions of avidin and nucleic acid, preferably both in salt-free water.
• the molar ratios of avidin to nucleobases NB is within the range from 0.44 to 0.0001 and preferably from 0.133 to 0.0044, and more preferably 0.044.
• the reagents are mixed under continuous stirring at a temperature from 0 to 50 Q C for a time between 1 and 600 seconds.
• the biotinylated surface protecting agent B-X 3 -PAi is prepared by synthesis or, if commercially available, is purchased.
• Preparation of B-X 3 -PAi by synthesis involves conjugating the biotin molecule to the polymer PAi, by chemical means, using classical bioconjugation techniques known to any expert of the art. Subsequently, the previously prepared or purchased biotinylated surface protecting agent B-X 3 -PAi, is added in a stoichiometrically controlled quantity relative to the concentration of biotin binding sites present in the solution, which are themselves relative to the avidin concentration.
• the molar ratios of avidin: B- X 3 -PAi are hence comprised between 4 and 0.02. Addition of the biotinylated surface protecting agent B-X 3 -PAi, is also carried out under stirring in aqueous solutions at a controlled temperature from 0 to 50 Q C for a time between 1 and 120 minutes.
• the nanoassembled complexes of the invention can be prepared by a method in which steps a) and b) are substantially inverted, hence the preparation method can comprise: a) adding the biotinylated surface protecting agent B-X 3 -PAi, to the avidin in predefined stoichiometric molar ratios of biotin to avidin; b) adding nucleic acid to the conjugate Av y (B-X 3 -PAi,) z obtained in the preceding step in pre-defined stoichiometric molar ratios of avidin to nucleobases.
• the preparation conditions are the same as those previously described for the first method.
• the preparation method can further comprise the purification of the particles from any monomeric avidin eventually present in the solution as a residue of the first step. Purification can be undertaken after either step a) or step b).
• Purification of the nanoassembled complexes from any monomeric avidin present in solution can be carried out by known methods, for example ultrafiltration or size exclusion chromatography.
• suitable systems are used, characterized by a cut-off equal to or greater than (>) 100 kDa.
• size exclusion chromatography chromatographic media are used which are suitable for retaining protein molecules of sizes up to ( ⁇ ) 200 kDa. If the biotin binding sites present on the avidin of the nucleus are not saturated by the biotinylated surface protecting agent B-X 3 -PAi, the preparation method can also comprise a further optional step of adding additional biotinylated compounds equal or different each other.
• nanoassembled complexes having the features of nanoparticles of any size can be obtained.
• said nanoassembled complexes are in form of nanoparticles of at least 10 nm in size and preferably from 50 to 1 ,000 nm in size.
• the use of nanoassembled complexes in nanoparticulate form herein described extends to all currently known applications of the avidin-biotin system, for which they act as "amplification" systems. Examples of these applications include their use as: a) detection means in in vitro diagnostics; b) amplifiers in the localization and patterning of molecules on surfaces (for example microarrays, protein chips and DNA); c) instruments for in vivo diagnostics; d) systems for active or passive targeting of drugs.
• biotinylated compounds which can further be introduced onto nanoassembly surfaces through biotin binding sites present on the avidin and not saturated by binding with biotin of the protecting agent B-X 3 - PAi,.
• the nanoassembly compounds obtained by the previously described preparation were characterized by: a) their size, using light scattering and electronic microscopy techniques; b) the degree of dispersion, using light scattering; c) the number of biotin binding sites available for introducing additional biotinylated functions.
• This assessment was carried out using the HABA assay, as described in the literature (Green NM Biochem. J. 1965, 94: 23C-24C); d) the speed of aggregation in a buffered medium, using light scattering techniques; e) their stability to freezing and thawing, and to lyophilization, using light scattering techniques.
• Example 1 Preparation and characterization of nanoassemblies obtained with plasmid DNA and avidin in different molar ratios without addition of a surface protector
• the complexes were prepared by mixing aqueous solutions of avidin (Av, Belovo,
• nucleic acid pNM, plasmid p-EGFP C1 (Clonetech#6084-1 ) (4.7
• the size distribution measured on the first sample is given in fig 1. From the figure said assembly can be seen to be characterized by a moderate polydispersivity.
• the data in table 1 also show that the sizes and polydispersivity of the nanoassembly increase as the y/n ratio decreases, indicating that as this value decreases, the degree of condensation of the nucleic acid molecule in the assembly is less.
• the size variation as y/n varies is however limited to within the values of about 70 to 200 nm.
• Example 2 Preparation and characterization of nanoassemblies obtained with plasmid DNA, avidin and surface protecting agents
• B-X 3 -PAi various surface protecting agents
• B-X 3 -PAi avidin molar ratios varying between 0 and 2.4 as shown in table 2.
• Five different B-X 3 -PAi, agents were used (I, Na, lib, III and IV), whose chemical formulas are given as follows:
• Said protecting agents B-X 3 -PAi were synthesized and characterized as described below.
• B-X ⁇ -PAh I was obtained by condensing the 6-amino-n-hexylamide of biotin with the N-hydroxysuccinimidyl carbonate of monomethoxy polyethylene glycol 2,000 (Monfardini C, Schiavon O et al. Bioconjugate Chemistry 1995, 6: 62-69).
• B-X ⁇ -PAh Na was obtained by condensing ⁇ -amino, co-methoxy-polyethylene glycol 5,000 (Fluka cat#06679) with the N-hydroxysuccinimidyl carbonate of biotinyl-n-hexanolamide (Morpurgo M, Bayer EA et al. J. Biochem. Biophys. Meth.
• B-X ⁇ -PAh Nb was obtained in a similar manner to B-X 3 -PAh I using monomethoxy polyethylene glycol 5,000 instead of 2,000.
• B-X 3 -PAh III was obtained by condensing the N-hydroxysuccinimidyl carbonate of monomethoxy polyethylene glycol 2,000 with the amino groups of the amide of 2,6 diaminohexanoic acid and with biotinyl-n-hexyldiamine (2,6-diamino-hexanoic acid
• B-X 3 -PAh IV was obtained in a similar manner to B-X 3 -PAh III using monomethoxy polyethylene glycol 5,000 instead of 2,000.
• Example 3 Preparation and characterization of nanoassemblies obtained with plasmid DNA and avidin, and purification by ultrafiltration.
• Alexa-Fluor546-biocytin (Molecular probes # A12923) was added to sample 1 Av- pEGFP 3, prepared as described in example 1 , in a quantity equal to that needed to saturate 2% of total biotin binding sites.
• the prepared product was subjected to various ultrafiltration steps using Vivaspin 100K PES membranes (Sartorius, 100,000 Da cut-off) so as to enable monomeric but not nanoassembled avidin to pass through.
• the avidin concentration in the supernatant and in the filtrate was determined by fluorescence, based on the signal of the Alexa-Fluor546 fluorophore.
• the supernatant obtained after four ultrafiltration steps was analyzed by light scattering.
• the avidin :NB ratio in the nanoparticulate system was calculated from the avidin concentration present therein with the assumption that the DNA present was the same as that present prior to ultrafiltration. Table 3. Composition of the solution containing nanoparticles before and after their purification expressed as avidin :nucleobase ratio (y/n)
• Example 4 Preparation and characterization of nanoassemblies obtained with genomic DNA and avidin in different molar ratios, with and without addition of surface protector.
• the nanocomplexes were prepared by mixing aqueous solutions of avidin (Av,
• Example 5 First comparison of efficiency of avidin in monomeric form and in nanocomplexed form with nucleic acid, in dot blot fluorescent detection
• a biotinylated antibody (anti-h PSMA) was immobilized by spotting (1 ⁇ l) onto nitrocellulose membranes. The membranes were blocked by immersing into PBS containing 2% w/v of BSA (PBS/BSA) then treated with solutions containing avidin
• Example 6 Second comparison of efficiency of avidin in monomeric form and in nanocomplexed form with nucleic acid, in dot blot fluorescent detection Varying quantities of biotinylated BSA (100, 50, 20, 10, 5, 2 ng of protein corresponding respectively to 10, 5, 2, 1 , 0.5 and 0.2 pmoles of biotin/spot) were immobilized by spotting (0.1 ⁇ l) onto nitrocellulose membranes.
• the membranes were blocked by immersing into PBS containing 2% w/v of BSA (PBS/BSA) then treated with solutions containing avidin (5 ⁇ g/ml in PBS/BSA), with previously added biotin-Alexa-Fluor® in a quantity so as to saturate 40% of total biotin binding sites.
• the avidin in said solutions was used in the monomeric or nanoassembly form (table 6). Table 6.
• the nanoassembly samples obtained with genomic DNA as given in example 4 were subjected to a freeze-thaw cycle.
• the size measurements of the particles present in solution after thawing were compared to those of the same preparations before treatment. The results are shown in table 7.
• the particles devoid of protection agent are not resistant to the freeze-thaw process, subsequent to which they aggregate irreversibly.
• the protecting agent B-X 3 -PAi is present on the surface, aggregation is inhibited.
• Example 8 Comparison of efficiency of avidin in monomeric form and in nanocomplexed form with nucleic acid, in an enzyme-linked detection system
• Varying quantities of biotinylated-lgG (IgG-B) (0.054, 0.18, 0.6, 2.0, 6.7 and 22.3 ng of protein were immobilized by spotting (0.5 ⁇ l) onto nitrocellulose membranes.
• the membranes were blocked by immersing into PBS containing 2% w/v of BSA (PBS/BSA) then treated with solutions containing avidin (5 ⁇ g/ml in PBS/BSA), The avidin in said solutions was used in the monomeric or nanoassembly form (table 7).

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